Compositions and methods for treating skin cancer associated diseases

ABSTRACT

The invention provides compositions and methods for treating skin cancer associated diseases. Specifically, the invention relates to topically administering a signaling pathway inhibitor or a related compound to treat pre-cancerous skin lesions, skin tumors, and their associated diseases or disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to International Patent ApplicationPCT/US 12/21732, filed Jan. 18, 2012, which claims priority to U.S.Provisional Patent Application 61/434,078, filed Jan. 19, 2011, each ofwhich is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates to compositions and methods for treating skincancer associated diseases. Specifically, the invention relates totopically administering tyrosine kinase inhibitors and related compoundsto treat pre-cancerous skin lesions, skin tumors, and their associateddiseases or disorders.

BACKGROUND OF THE INVENTION

Actinic keratosis (AK) is the most common precancerouslesion—manifesting as thickened scaly or crusty patches of skin—inhumans, and its treatment represents a common reason for patients to seea dermatologist. Recent studies estimate that there are approximately 58million AKs in the US population leading to 5.2 million office visitsper year with an approximate cost of 0.9-1.2 billion dollars. The riskfor developing an AK is directly related to the degree of photodamagewhich is a function of age, sun exposure, and Fitzpatrick-skin type(being more common in fair-skinned people). Studies show that multipleAKs significantly decrease the quality of life in patients, leading someinvestigators to use the term ‘actinic neoplasia syndrome’ for suchpatients. Some of these pre-cancers progress to squamous cell carcinoma(cSCC), which is discussed below.

Current AK treatments involve topical and destructive modalities. AKscan be treated by freezing the skin with liquid nitrogen (N₂). Althoughtechnically simple, this therapy has adverse effects which includedyspigmentation, pain, stinging, and a low efficacy rate; liquid N₂ isassociated with only an 83% clearance rate and lacks specificity forlesional cells requiring the freezing of at least 1 mm of surroundingnon-lesional tissue. Other commonly used destructive therapies include:curettage and surgical excision. Since patients often have many AKsrequiring treatment; this limits the use of destructive modalitiesbecause of the cosmetic deficits associated with dyspigmentation andscarring.

The primary topical agents to treat AKs are 5-fluorouracil (5-FU),imiquimod (ALDARA®), and diclofenac (e.g., SOLARAZE®) 5-FU is achemotherapeutic agent that inhibits RNA and DNA synthesis and targetsdividing cells which are more prevalent in AKs than adjacent epidermis.5-FU is used as 0.5%, 2.5%, or 5.0% formulations which correspond tocompound concentrations of (36-360 mM). 5-FU has significant sideeffects including prominent inflammation, ulceration, and even scarring;given these side-effects, topical 5-FU is associated with a lowerquality of life for patients. Imiquimod, a Toll receptor 7 agonist,produces prominent inflammation to eliminate AKs. The prominentinflammation stimulated by imiquimod is irritating and mimics psoriasis,and this reaction has been associated with autoimmune reactions such asalopecia areata and vitiligo. Another common topical agent to treat AKsis diclofenac, a topical non-steroidal anti-inflammatory compound withan unclear mechanism of action on actinic keratoses. Recent studiesusing diclofenac and imiquimod showed therapeutic weaknesses associatedwith poor clearance rates and significant irritation. Overall, the mostcommonly used topical agents to treat AKs have significant negativefeatures including irritation, decreased quality of life, and limitedefficacy.

cSCC is the second most common form of cancer with an annual incidenceof 620,000 cases annually. According to some studies, cSCCs causeapproximately 8,000 cases of nodal metastasis leading to 3,000 deaths inthe US alone each year. In 2004, the estimated cost of treating cSCCs inthe US alone was approximately 500 million dollars. The risk factors forcSCCs are similar to those for AKs and include age, male sex, andcumulative UV exposure from sunlight or tanning salons. Since Americansare living longer, spending more time outdoors, and not adequately usingsunscreens, it is likely that the numbers of cSCCs in the United Stateswill increase, as will the cost to treat them.

Accordingly, there exists a need for improved therapy modalities andcompositions to treat pre-cancerous skin lesions, skin tumors, and theirassociated diseases or disorders.

SUMMARY OF THE INVENTION

In one aspect, methods are provided for treating a pre-cancerous skinlesion in a subject, the methods include: topically administering tosaid subject a therapeutically effective amount of a tyrosine kinaseinhibitor (e.g., an Src family kinase inhibitor) or related compound,thereby treating said skin lesion in said subject.

In another aspect, methods are provided for treating a skin tumor in asubject, the methods include: topically administering to said subject atherapeutically effective amount of a tyrosine kinase inhibitor (e.g.,an Src family kinase inhibitor) or related compound, thereby treatingsaid skin tumor in said subject.

In a further aspect, methods are provided for treating an actinickeratosis in a subject, the methods include: topically administering tosaid subject a therapeutically effective amount of a tyrosine kinaseinhibitor (e.g., an Src family kinase inhibitor) or related compound,thereby treating said actinic keratosis in said subject.

In yet another aspect, methods are provided for treating a cutaneoussquamous cell carcinoma in a subject, the methods include: topicallyadministering to said subject a therapeutically effective amount of atyrosine kinase inhibitor (e.g., an Src family kinase inhibitor) orrelated compound.

In yet another aspect, compositions are provided, the compositionsinclude: a topical formulation of a tyrosine kinase inhibitor or relatedcompound present in an amount effective to topically treat a skin cancerrelated disease or disorder (e.g., a pre-cancerous skin lesion, a skintumor, an actinic keratosis, a squamous cell carcinoma in situ likelesion and/or a cutaneous squamous cell carcinoma).

In yet another aspect, methods are provided for treating a skin cancerrelated disease or disorder (e.g., a pre-cancerous skin lesion, a skintumor, an actinic keratosis, a squamous cell carcinoma in situ likelesion and/or a cutaneous squamous cell carcinoma) in a subject, themethods include: topically administering to said subject atherapeutically effective amount of a PI3K/PDK1/AKT signaling pathwayinhibitor.

In yet another aspect, compositions are provided, the compositionsinclude: a topical formulation of a PI3K/PDK1/AKT signaling pathwayinhibitor present in an amount effective to topically treat a skincancer related disease or disorder (e.g., a pre-cancerous skin lesion, askin tumor, an actinic keratosis, a squamous cell carcinoma in situ likelesion and/or a cutaneous squamous cell carcinoma).

In yet another aspect, methods are provided for treating a skin cancerrelated disease or disorder (e.g., a pre-cancerous skin lesion, a skintumor, an actinic keratosis, a squamous cell carcinoma in situ likelesion and/or a cutaneous squamous cell carcinoma) in a subject, themethods include: topically administering to said subject atherapeutically effective amount of a MAPK/ERK signaling pathwayinhibitor.

In yet another aspect, compositions are provided, the compositionsinclude: a topical formulation of a MAPK/ERK signaling pathway inhibitorpresent in an amount effective to topically treat a skin cancer relateddisease or disorder (e.g., a pre-cancerous skin lesion, a skin tumor, anactinic keratosis, a squamous cell carcinoma in situ like lesion and/ora cutaneous squamous cell carcinoma).

In yet another aspect, methods are provided for treating a skin cancerrelated disease or disorder (e.g., a pre-cancerous skin lesion, a skintumor, an actinic keratosis, a squamous cell carcinoma in situ likelesion and/or a cutaneous squamous cell carcinoma) in a subject, themethods include: topically administering to said subject atherapeutically effective amount of a JAK-STAT signaling pathwayinhibitor.

In yet a further aspect, compositions are provided, the compositionsinclude: a topical formulation of a JAK-STAT signaling pathway inhibitorpresent in an amount effective to topically treat a skin cancer relateddisease or disorder (e.g., a pre-cancerous skin lesion, a skin tumor, anactinic keratosis, a squamous cell carcinoma in situ like lesion and/ora cutaneous squamous cell carcinoma).

Other features and advantages of the present invention will becomeapparent from the following detailed description examples and figures.It should be understood, however, that the detailed description and thespecific examples while indicating preferred embodiments of theinvention are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription. It is also contemplated that whenever appropriate, anyembodiment of the present invention can be combined with one or moreother embodiments of the present invention, even though the embodimentsare described under different aspects of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Signaling model. Arrows indicate positive regulation. T-barsindicate inhibition. Fyn increases the activity of Erk 1/2, STAT-3, andPDK-1. Fyn inhibits PKCδ and p53. Srcasm negatively regulates Fyn. Rasinduces Fyn mRNA levels through an Akt-dependent mechanism. PDK-1activates Akt.

FIG. 2 demonstrates the inverse relationship between SFK activity andSrcasm levels in AKs Immunohistochemistry for activated SFKs and Srcasmshows an inverse relationship between SFK activity and Srcasm in AKs andunremarkable epidermis (Unr).

FIG. 3. K14 Fyn Y528F mice demonstrate prominent epidermal hyperplasiaafter acute UVB exposure. Mice (n=3) of the indicated genotypes weresubjected to one dose of 1200 mJ/cm² UVB. Prominent epidermalhyperplasia was noted in K14-Fyn Y528F mice. Note that increasing Srcasmlevels corrects this UV-induced hyperplasia in the K14-FynY528F/K14-Srcasm double transgenic. H+E, 100×

FIG. 4. K14 Fyn Y528F mice demonstrate enhanced cSCC formation after 7weeks of UVB exposure. Six week old mice (2 per cohort) were subjectedto UVB on Monday-Wednesday-Friday at 300 mJ/cm² first week, 400 mJ/cm²second week, and 500 mJ/cm² subsequent weeks. New tumor formation(average 4 per mouse) was seen only in K14-Fyn Y528F mice. Arrowsindicate cSCCs arising after UVB treatment. The other tumors arosebefore UVB.

FIG. 5. Ki-67 staining demonstrates prominent UVB-inducedhyperproliferation in K14-Fyn Y528F mice. Ki-67 staining of biopsiesshown in FIG. 3 demonstrates increased nuclear staining of the basal andsuprabasal layers in K14-Fyn Y528F mice. Sparse nuclear positivity wasseen in day 0 (pre-UV) biopsies and day 10 of control mice. 200×

FIG. 6. K14-Fyn Y528F mice demonstrate persistent H2A.X phosphorylation.Immunohistochemical staining for phospho-H2A.X demonstrates subnuclearpositivity (arrows) in K14-Fyn Y528F mice at day 10. No staining wasdetected in day 10 control or Fyn/Srcasm double transgenic mice. 400×

FIG. 7. Topical PP2 induces clearance of a cSCC in K14 Fyn Y528F mouse.One millimolar PP2 in DMSO (25 μL) was topically applied once a dayMonday-Friday for 30 times. Clearance of a large facial cSCC was noted(top panels). A PP3 (inactive control compound) treated lesion on thesame mouse remained stable (bottom panels).

FIG. 8. Topical PP2 induces clearance of cSCC in K14 Fyn Y528F mice asearly as 21 days. One millimolar PP2 in DMSO was topically applied oncea day Monday-Saturday for 20 times. Clearance of a large facial cSCC wasnoted by 21 days.

FIG. 9. Histologic features of PP2-induced clearance of cSCC in K14 FynY528F mice. One millimolar PP2 in DMSO was topically applied once a dayMonday-Friday for 30 times to a cSCC covering the dorsum of the ear.Prominent clearance of the cSCC was noted six weeks after beginningtreatment. The open rectangle designates the region of the ear taken forhistology. Small black rectangle=1 mm

FIG. 10. Clearance of precancerous lesion and a cSCC by PP2 in threeweeks. One millimolar PP2 in DMSO was topically applied once a dayMonday-Friday for 20 times to a small precancerous lesion on the ear andto a cSCC on the neck. Clearance of both lesions was noted by threeweeks after initiating treatment. Small black rectangle=2 mm

FIG. 11 shows that topical PP2 induces tumor regression in five weeks.

FIG. 12 shows that topical dasatinib reduces cSCC size in cohort study.11 K14-Fyn Y528F mice with solitary cSCCs were given topical 1%dasatinib ointment daily (Monday-Friday) for 25 applications. A controlcohort of 6 mice was treated in parallel with vehicle. The area of eachtumor was followed over time and the relative area remaining for eachtumor was calculated by comparing the area at each time point to thearea at time 0. The average relative tumor area value was calculated foreach cohort at the indicated times with the standard error. p valueswere calculated using a T-test for means.

FIG. 13 shows that topical dasatinib induces clearance of cSCCs inK14-Fyn Y528F mice. 1% dasatinib (DST) ointment was topically applieddaily (Monday-Friday) for 25 applications-numbers indicate treatments.Clearance of a back cSCC was noted by 5 weeks. Application of vehicle(Con) did not clear a cSCC of similar size during the treatment period.Images were taken with dermatoscope under identical conditions.

FIG. 14 shows that topical BEZ235 induces cSCC regression. FifteenK14-Fyn Y528F mice with solitary cSCCs were given topical 20 mM BEZ235ointment daily (Monday-Friday) for 25 applications. A control cohort of15 mice was treated in parallel with ointment alone. The area of eachtumor was followed over time and the relative area for each tumor wascalculated by comparing the area at each time point to the area at time0. The average relative tumor area value was calculated for each cohortat the indicated times with the standard error. p values were calculatedusing a T-test for means.

FIG. 15 shows that phosphorylation of S6 kinase, an mTOR substrate, iselevated in K14-Fyn Y528F mice in cSCCs (right panel) as compared tounremarkable tissue (left panel).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to compositions and methods for treating skincancer associated diseases. Specifically, the invention relates totopically administering tyrosine kinase inhibitors or related compoundsto treat pre-cancerous skin lesions, skin tumors, and their associateddiseases or disorders, which include without limitation, actinickeratosis (AK), squamous cell carcinoma in situ like lesion (SCIS-likelesion) and cutaneous squamous cell carcinoma (cSCC).

In one embodiment, provided herein are methods for treating apre-cancerous skin lesion in a subject, the methods comprising:topically administering to said subject a therapeutically effectiveamount of a tyrosine kinase inhibitor or related compound, therebytreating said skin lesion in said subject. In another embodiment,provided herein are methods for treating a skin tumor in a subject, themethods comprising: topically administering to said subject atherapeutically effective amount of a tyrosine kinase inhibitor orrelated compound, thereby treating said skin lesion in said subject.

In another embodiment, provided herein are methods for treating anactinic keratosis in a subject, the methods comprising: topicallyadministering to said subject a therapeutically effective amount of atyrosine kinase inhibitor or related compound, thereby treating saidactinic keratosis in said subject. In another embodiment, providedherein are methods for treating cutaneous squamous cell carcinoma in asubject, the methods comprising: topically administering to said subjecta therapeutically effective amount of a tyrosine kinase inhibitor orrelated compound, thereby treating said cutaneous squamous cellcarcinoma in said subject.

The Applicants have surprisingly and unexpectedly found thatadministering tyrosine kinase inhibitors (e.g., an Src family kinaseinhibitor) and related compounds treat pre-cancerous skin lesions, skintumors, and their associated diseases or disorders. For example,topically administering PP2, dasatinib and BEZ235 surprisingly andunexpectedly induced the clearance of cutaneous squamous cell carcinomaand pre-cancerous lesions within a few weeks, as demonstrated herein.

The term “tyrosine kinase inhibitor” may refer to any of a variety oftherapeutic agents or drugs that act as selective or non-selectiveinhibitors of receptor and/or non-receptor tyrosine kinases. Tyrosinekinase inhibitors and related compounds are well known in the art anddescribed in U.S Patent Publication 2007/0254295, which is incorporatedby reference herein in its entirety. It will be appreciated by one ofskill in the art that a compound related to a tyrosine kinase inhibitorwill recapitulate the effect of the tyrosine kinase inhibitor, e.g., therelated compound will act on a different member of the tyrosine kinasesignaling pathway to produce the same effect as would a tyrosine kinaseinhibitor of that tyrosine kinase.

Examples of tyrosine kinase inhibitors and related compounds suitablefor use in methods of embodiments of the present invention include, butare not limited to, dasatinib (BMS-354825), PP2, BEZ235, saracatinib,gefitinib (Iressa), sunitinib (Sutent; SU11248), erlotinib (Tarceva;OSI-1774), lapatinib (GW572016; GW2016), canertinib (CI 1033), semaxinib(SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib(Gleevec; STI571), leflunomide (SU101), vandetanib (Zactima; ZD6474),MK-2206(8-[4-aminocyclobutyl)phenyl]-9-phenyl-1,2,4-triazolo[3,4-f][1,6]naphthyridin-3(2H)-onehydrochloride) derivatives thereof, analogs thereof, and combinationsthereof. Additional tyrosine kinase inhibitors and related compoundssuitable for use in the present invention are described in, for example,U.S Patent Publication 2007/0254295, U.S. Pat. Nos. 5,618,829,5,639,757, 5,728,868, 5,804,396, 6,100,254, 6,127,374, 6,245,759,6,306,874, 6,313,138, 6,316,444, 6,329,380, 6,344,459, 6,420,382,6,479,512, 6,498,165, 6,544,988, 6,562,818, 6,586,423, 6,586,424,6,740,665, 6,794,393, 6,875,767, 6,927,293, and 6,958,340, all of whichare incorporated by reference herein in their entirety.

In certain embodiments, the tyrosine kinase inhibitor is an Src familykinase (SFK) inhibitor.

In certain embodiments, the tyrosine kinase inhibitor or relatedcompound has a molecular weight of less than 500 daltons.

In certain embodiments, the tyrosine kinase inhibitor is a smallmolecule kinase 2 0 inhibitor that has been orally administered and thathas been the subject of at least one Phase I clinical trial, morepreferably at least one Phase II clinical, even more preferably at leastone Phase III clinical trial, and most preferably approved by the FDAfor at least one hematological or oncological indication. Examples ofsuch inhibitors include, but are not limited to, Gefitinib, Erlotinib,Lapatinib, Canertinib, BMS-599626 (AC-480), Neratinib, KRN-633,CEP-11981, Imatinib, Nilotinib, Dasatinib, AZM-475271, CP-724714,TAK-165, Sunitinib, Vatalanib, CP-547632, Vandetanib, Bosutinib,Lestaurtinib, Tandutinib, Midostaurin, Enzastaurin, AEE-788, Pazopanib,Axitinib, Motasenib, OSI-930, Cediranib, KRN-951, Dovitinib, Seliciclib,SNS-032, PD-0332991, MKC-I (Ro-317453; R-440), Sorafenib, ABT-869,Brivanib (BMS-582664), SU-14813, Telatinib, SU-6668, (TSU-68), L-21649,MLN-8054, AEW-541, and PD-0325901.

Also provided herein are compositions comprising: a formulation fortopical administration of a tyrosine kinase inhibitor (e.g., Src familykinase (SFK) inhibitor) or related compound amount effective totopically treat a skin cancer related disease or disorder. Src familykinases are well known in the art and described in U.S. PatentPublication 2008/0193504, which is incorporated by reference herein inits entirety. Members of the Src family include, for example, thefollowing kinases in mammals: Src, Fyn, Yes, Fgr, Lyn, Hck, Lck, andBlk. These are nonreceptor protein kinases that range in molecular massfrom about 52 Kd to about 62 Kd. All are characterized by a commonstructural organization having six distinct functional domains: Srchomology domain 4 (SH4), a unique domain, SH3 domain, SH2 domain, acatalytic domain (SH1), and a C-terminal regulatory region. See Tatosyanet al. Biochemistry (Moscow) 65, 49-58 (2000).

As used herein, the terms “Src family tyrosine kinase inhibitor” “Srcfamily kinase inhibitor” or “SFK inhibitor” may refer to any inhibitorof one or more kinases within the Src family of kinases.

SFK inhibitors are well known in the art and described in U.S. PatentPublication 2008/0193504, which is incorporated by reference herein inits entirety. In one embodiment, the SFK inhibitor PP2(4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine;

also known as AG1879; Calbiochem MW0301.8) is used. PP2 is alsodescribed in J. H. Hanke et al., 1996, J. Biol. Chem, vol. 271, page695; J. K. Chen et al., 2000, J. Biol. Chem, vol. 275, page 13789; andYoshizumi et al., 2000, J. Biol. Chem., vol. 275, page 11706. In anotherembodiment the SFK inhibitor PP1 (Molecular Cell, 1999, 3: 639-648) isused. In another embodiment the SFK inhibitor SKI606 (Cancer Research,2003, 63: 375) is used. In another embodiment, the SFK inhibitor AZD0530(AstraZeneca, London UK) is used. In another embodiment, the SFKinhibitor SU6656 is used (Blake, R. A. et al. 2000. Mol. Cell. Biol. 20:9018-9027). In another embodiment the SFK inhibitor dasatinib(BMS-354825)(N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazolecarboxamide,monohydrate) is used.

SFK inhibitors useful in the methods of the present invention alsoinclude, but are not limited to, SFK inhibitors described inInternational Patent Applications WO 96/10028; WO 97/07131; WO 97/08193;WO 97/16452; WO 97/28161; WO 97/32879; WO 97/49706; WO 01/94341; WO02/16352; WO 02/30924; WO 02/30926; WO 02/34744; WO 02/085895; WO 3 002/092577; WO 02/092578; WO 02/092579; WO 03/008409 and WO 03/013540,all of which are incorporated by reference herein in their entirety.

Other examples of SFK inhibitors include, but are not limited to,4-amino-5-(3-methoxyphenyl)-7-{4-[2-(2-methoxyethylamino)ethoxy]phenyl}pyrrolo[2,3-d]pyrimidineand 4-amino-5-(3-methoxyphenyl)7-(4-{2-[di-(2-methoxyethyl)amino]ethoxy}phenyl)pyrrolo[2,3-d]pyrimidine which are obtainable by methods described inInternational Patent Application WO 96/10028;2-(2,6-dichloroanilino)-6,7-dimethyl-1,8-dihydroimidazo[4,5-h]isoquinolin-9-oneand 2-(2,6-dichloroanilino)-7-[(E)-3-diethylaminoprop-1-enyl]-6-methyl1,8-dihydroimidazo[4,5-h]isoquinolin-9-one which are obtainable bymethods described in Journal Medicinal Chemistry, 2002, 45: 3394;1-[6-(2,6-dichlorophenyl)-2-(4-diethylaminobutyl)pyrido[2,3-d]pyrimidin-7-yl]3-ethylureawhich is obtainable by methods described in Journal Medicinal Chemistry,1997, 40: 2296-2303 and Journal Medicinal Chemistry, 2001, 44, 1915;certain 4-anilino-3-cyanoquinoline derivatives (Journal MedicinalChemistry, 2001, 44: 822-833 and 3965-3977); PD166285 (J. Pharmacol.Exp. Ther., 1997, 283: 1433-1444); PD162531 (Mol. Biol. Cell, 2000, 11:51-64); PD166326 and PD180970 (Biochem. Pharmacol., 2000, 60: 885-898);PD173955 (Cancer Research, 1999, 59; 6145-6152); PD173952 (Blake, R. A.et al. 2000. Mol. Cell. Biol. 20: 9018-9027); Dasatinib or BMS354825(Lombardo et al. 2004. J Med. Chem. 47:6658-6661); thepyrazolopyrimidine class of SFK inhibitors, such as4-amino-5-(4-methylphenyl)-7-(t-butyl-) pyrazolo[3,4-d-]pyrimidine (AGL1872), 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d-]pyrimidine(AGL 1879), and the like; the macrocyclic dienone class of SFKinhibitors, such as Radicicol R2146, Geldanamycin, Herbimycin A, and thelike; the pyrido[2,3-d]pyrimidine class of SFK inhibitors; the4-anilino-3-quinolinecarbonitrile class of SFK inhibitors;tyrphostin-derived inhibitors, which are derivatives ofbenzylidenemalonitrile (Ramdas et al., 1995, Archives of Biochemistryand Biophysics 323:237-242); derivatives of pyrazolopyrimidine PP1(4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (Hankeet al., 1996, J. Biol. Chem. 271:695-791); angelmicin B and derivativesthereof (Yokoyama et al., 1996, Leukemia Research 20:491-497); compoundsdescribed in J Bone Mineral Research, 1999, 14: (Suppl. 1) S487,Molecular Cell, 1999, 3: 639-647, Journal Medicinal Chemistry, 1997, 40:2296-2303, Journal Medicinal Chemistry, 1998, 41: 3276-3292 andBioorganic & Medicinal Chemistry Letters, 2002, 12: 1361 and 3153.

Additional SFK inhibitors useful in the methods the present inventionare described in, for example, International Patent Applications WO05/013983, WO 02/079192, WO 03/000188, WO 03/000266, WO 03/000705, WO02/083668, WO 02/092573, WO 03/004492, WO 00/49018, WO 03/013541, WO01/00207, WO 01/00213, WO 01/00214 and U.S. Patent Applications2005/0096298, 2004/0266855, 2004/0204582, 2004/0167198, 2004/0014676,2003/0207902, 2003/0119819 and 2002/0132819, all of which areincorporated by reference in their entirety.

In another embodiment, as described in U.S. Patent Publication2008/0193504, small peptides which compete with larger phosphotyrosinepeptides for binding to the Src kinase protein may be used to inhibitSrc family kinases, in particular small phosphotyrosine containingpeptide ligands, 5 to 6 amino acids, which are able to compete withlarger phosphotyrosine-containing peptides and protein ligands forbinding to SH2 domains, thereby inhibiting the Src family kinases. Inanother embodiment, small peptides which correspond to catalytic orenzymatic domains of Src kinase and would compete with Src kinase may beused to inhibit the activation of downstream components of the Srckinase signaling cascade. In another embodiment, SFK inhibitors includethe use of larger polypeptides that inhibit Src kinase activityincluding, but not limited to, Csk (carboxyl-terminal Src kinase) whichis a specific physiologic inhibitor of Src kinase. Further examples oflarger polypeptides that inhibit Src kinase activity include, forexample, Src dominant-negative mutants, i.e., Srck-(Barone et al., 1995,Nature 378:509-512) and Fyn dominant-negative mutants (Twamley-Stein etal., 1993, Proc. Natl. Acad. Sci. USA 90:7696-7700). In someembodiments, antibodies or other larger molecules that inhibit Srckinase activity may be used.

As used herein, the term, “selective” with respect to inhibition meanspreferential inhibition of a first activity relative to a secondactivity (e.g., preferential inhibition of one signaling pathway toanother signaling pathway; preferential inhibition relative to othertyrosine kinase inhibitors; or preferential inhibition of a mutant to awild-type or vice versa). In some embodiments, the inhibitor is greaterthan five times more selective, greater than ten times more selective,greater than fifty times more selective, greater than 100 times moreselective, or greater than 1000 times more selective for the desiredmolecular target or pathway versus an undesired molecular target orpathway. In some embodiments, the inhibitor inhibits the first activityof the molecular target or pathway by at least 2-fold, at least 5-fold,at least 10-fold, at least 20-fold, at least 50-fold, at least 100-foldrelative to the second activity of the molecular target or pathway underthe same conditions. The activity of a molecular target or pathway maybe measured by any reproducible means. The activity of a moleculartarget or pathway may be measured in vitro or in vivo.

As used herein, “modulating” refers to “stimulating” or “inhibiting” anactivity of a molecular target or pathway. In one aspect, a compositionmodulates the activity of a molecular target or pathway if it stimulatesor inhibits the activity of the molecular target or pathway by at least10%, by at least about 10%, by at least about 20%, by at least about25%, by at least about 30%, by at least about 40%, by at least about50%, by at least about 60%, by at least about 70%, by at least about75%, by at least about 80%, by at least about 90%, by at least about95%, by at least about 98%, or by about 99% or more relative to theactivity of the molecular target or pathway under the same conditionsbut lacking only the presence of the composition. In one aspect, thecomposition modulates the activity of a molecular target or pathway ifit stimulates or inhibits the activity of the molecular target orpathway by at least 2-fold, at least 5-fold, at least 10-fold, at least20-fold, at least 50-fold, at least 100-fold relative to the activity ofthe molecular target or pathway under the same conditions but lackingonly the presence of the composition. The activity of a molecular targetor pathway may be measured by any reproducible means. The activity of amolecular target or pathway may be measured in vitro or in vivo. Forexample, the activity of a molecular target or pathway may be measuredin vitro or in vivo by an enzymatic activity assay. For example, samplesor assays comprising one or more components of a protein kinasesignaling pathway, such as one or more components of a PI3K/PDK1/AKTsignaling pathway (e.g., mTOR), can be treated with the composition andcompared to control samples without the composition. Control samples(untreated with the composition) can be assigned a relative activityvalue of 100%. A change in enzymatic activity caused by the compositioncan be measured in the assays. For example, the change in enzymaticactivity can be characterized by the change in the extent ofphosphorylation of certain substrates.

It will be appreciated that many of the inhibitors described herein arestrong inhibitors of their targets. For example, an inhibitor has abinding inhibitory activity (IC₅₀ value) for its desired moleculartarget of 1000 μM or less, 1000 nM or less, 100 nM or less, 10 nM orless, or especially 1 nM or less. In another example, the inhibitor hasa binding inhibitory activity (IC₅₀ value) for its desired moleculartarget of between 1000 μM and 1 nM, between 1000 μM and 10 nM, between1000 μM and 100 nM, between 1000 μM and 1000 nM, between 1000 nM and 1nM, between 1000 nM and 10 nM, between 1000 nM and 100 nM, between 100nM and 10 nM, between 100 nM and 1 nM, or between 10 nM and 1 nM.

In some embodiments, the inhibitors disclosed herein inhibit theirmolecular targets or pathways by at least about 10%, by at least about20%, by at least about 25%, by at least about 30%, by at least about40%, by at least about 50%, by at least about 60%, by at least about70%, by at least about 75%, by at least about 80%, by at least about90%, by at least about 95%, by at least about 98%, or by about 99% ormore.

As used herein,“phosphorylation” refers to the addition of phosphategroups to a substrate, including proteins and organic molecules; and,plays an important role in regulating the biological activities ofproteins. In one aspect, the phosphorylation assayed and measuredinvolves the addition of phosphate groups to tyrosine residues. Thesubstrate can be a peptide or protein.

The Janus kinases (JAK) are a family of intracellular non-receptortyrosine kinases that include JAK1, JAK2, JAK3 and tyrosine kinase 2(TYK2). The JAKs play a role in cytokine signaling. The downstreamsubstrates of the JAK family of kinases include the signal tranduceractivator of transcription (STAT) proteins. JAK/STAT signaling has beenimplicated in the mediation of many abnormal immune responses such asallergies, asthma, autoimmune diseases such as transplant (allograft)rejection, rheumatoid arthritis, amyotrophic lateral sclerosis andmultiple sclerosis, as well as in solid and hematologic malignanciessuch as leukemia and lymphomas. For a review of the pharmaceuticalintervention of the JAK/STAT pathway see Frank, 1999, Mol. Med.5:432:456 and Seidel et al., 2000, Oncogene 19:2645-2656.

Inhibitors of JAK/STAT include, but are not limited to, Ruxolitinib,Tofacitinib (tasocitinib; CP-690550), Baricitinib (LY3009104,INCB28050), CYT387, Lestaurtinib, Pacritinib (SB1518), TG101348.

The PI3K//PDK1/AKT/mTOR pathway is an intracellular signaling pathwayimportant in apoptosis. Briefly, PI3K activation leads to activation ofAKT which activates mammalian target of rapamycin (mTOR).

Inhibitors of PI3K include, but are not limited to, Wortmannin (anirreversible inhibitor of PI3K), demethoxyviridin (a derivative ofwortmannin), LY294002 (a reversible inhibitor of PI3K), Perifosine,CAL101, PX-866, IPI-145, SF1126, INK1117, GDC-0941, BKM120, XL147 (alsoknown as SAR245408), XL765 (also known as SAR245409), Palomid 529,GSK1059615, ZSTK474, PWT33597 (inhibits both PI3K and mTOR inhibitor),IC87114, TG100-115, CAL263, PI-103 (inhibits both PI3K and mTORinhibitor), GNE-477 (inhibits both PI3K and mTOR inhibitor), CUDC-907(inhibits both PI3K and mTOR inhibitor), AEZS-136, (also inhibitsErk1/2). AKT inhibitors include, but are not limited to, Miltefosine,VQD-002 and Perifosine.

mTOR inhibitors include, but are not limited to, ABT-578, AP-23675,AP-23573, AP-23841, CCI-779, temsirolimus, everolimus, Ridaforolimus,INK128, AZD8055, and AZD2014.

Inhibitors that inhibit both PI3K and mTOR include, but are not limitedto, BEZ235, BGT226, SF1126, PKI-587.

In the MEK/ERK pathway, activated Ras activates the protein kinaseactivity of RAF kinase. RAF kinase phosphorylates and activates MEK(MEK1 and MEK2). MEK phosphorylates and activates a mitogen-activatedprotein kinase (MAPK, and originally called ERK). RAF inhibitorsinclude, but are not limited to, SB590885, PLX4720, XL281, RAF265,LGX818, Regorafenib, RAF265, XL281, GDC0879, PLX4720, sorafenib andvemurafenib. MAPK inhibitors include, but are not limited to, XL518,CI-1040, PD035901, MEK162, selumetinib, Trametinib (GSK1120212).

Methods for making tyrosine kinase inhibitors and related compounds arewell known in the art, as described above.

In another embodiment, provided herein are pharmaceutical compositionsto treat a pre-cancerous skin lesions, skin tumors, and their associateddiseases or disorders in a subject, comprising: a therapeuticallyeffective amount of a tyrosine kinase inhibitor, wherein said tyrosinekinase inhibitor or related compound is present in an amount effectiveto treat said skin lesions, skin tumors, or their associated diseases ordisorders.

The pharmaceutical compositions described herein comprise thetherapeutic agents of this invention and one or more pharmaceuticallyacceptable carriers. “Pharmaceutically acceptable carriers” include anyexcipient which is nontoxic to the cell or mammal being exposed theretoat the dosages and concentrations employed. The pharmaceuticalcompositions may include one or more additional therapeutic agents.

According to certain embodiments, the pharmaceutical composition is agel or ointment having the topical formulation shown in Table 1 below.

TABLE 1 Component Percentage (%) Tyrosine kinase inhibitor(s) 0.5-1.5and related compounds ethanol or DMSO 7-9 propyleneglycol 17-19hydroxypropylmethylcellulose 1-2 Water 70-73

Pharmaceutically acceptable carriers include solvents, dispersion media,buffers, coatings, antibacterial and antifungal agents, wetting agents,preservatives, buffers, chelating agents, antioxidants, isotonic agentsand absorption delaying agents.

Pharmaceutically acceptable carriers include, for example, water;saline; phosphate buffered saline; dextrose; glycerol; alcohols such asethanol and isopropanol; phosphate, citrate and other organic acids;ascorbic acid; low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; EDTA; salt forming counterions such assodium; and/or nonionic surfactants such as TWEEN, polyethylene glycol(PEG), and PLURONICS; isotonic agents such as sugars, polyalcohols suchas mannitol and sorbitol, and sodium chloride; as well as combinationsthereof. Antibacterial and antifungal agents include parabens,chlorobutanol, phenol, ascorbic acid, and thimerosal.

For topical administration, embodiments of the invention may beformulated in the form of a lotion, cream, serum, spray, aerosol, cake,ointment, essence, gel, paste, patch, pencil, towelette, mask, stick,foam, elixir, concentrate, and the like form. Composition providedherein may be formulated as an immediate, controlled, extended ordelayed release composition. Preparations for topical administrationinclude aqueous or non-aqueous solutions, suspensions, emulsions, andgel. Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. In the subject invention, pharmaceutically acceptable carriersinclude, but are not limited to, 0.01-0.1 M and preferably 0.05 Mphosphate buffer or 0.8% saline. Other common vehicles include sodiumphosphate solutions, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. Other vehicles include fluid andnutrient replenishers, electrolyte replenishers, such as those based onRinger's dextrose, and the like. Preservatives and other additives mayalso be present such as for example, antimicrobials, antioxidants,chelating agents, and inert gases and the like. Suitable formulationsfor use in the therapeutic methods disclosed herein are described inRemington's Pharmaceutical Sciences, Mack Publishing Co., 16^(th) ed.(1980).

In some embodiments, the composition includes isotonic agents, forexample, sugars, polyalcohols, such as mannitol, sorbitol, or sodiumchloride. Prolonged absorption of the compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

The pharmaceutical compositions according to some embodiments of thepresent invention may further include a variety of pharmaceuticalingredients, which are added in order to fine-tune the consistency ofthe formulation, protect the formulation components from degradation andoxidation and bestow their cosmetic acceptability. Such excipients maybe selected from the group consisting of water, surfactants,emulsifiers, diglycerides, triglycerides, stabilizing agents,antioxidants, glycerol, ethanol, propanol, isopropanol, butanol,polymeric gelling agents, flavoring, colorant and odorant agents andother formulation components, used in the art of pharmaceutical andcosmetic formulary.

Additional active and inactive ingredients may also include, withoutlimitation, local analgesics such as benzocaine, menthol, and the like(wherein menthol is also capable of providing a soothing, coolingsensation), as well as, antihistamines and thickeners other than thosealready listed.

The compositions of the present invention can also include an emollient.Emollient is used to smooth the surface of the skin. Examples ofsuitable emollients include, but are not limited to, volatile andnon-volatile silicone oils (e.g., dimethicone, cyclomethicone,dimethiconol, and the like), highly branched hydrocarbons, and mixturesthereof. Emollients useful in the instant invention are furtherdescribed in U.S. Pat. No. 4,919,934, to Deckner et al., which isincorporated herein by reference in its entirety.

A variety of additional ingredients can be incorporated into thecomposition of the present invention. Non-limiting examples of theseadditional ingredients include vitamins and derivatives thereof (e.g.,tocopherol, panthenol, and the like); other thickening agents; saturatedand/or unsaturated alkyl alpha hydroxy acids; resins; gums (e.g., guargum, xantham gum and the like); waxes (both naturally occurring andsynthetic); polymers for aiding the film-forming properties andsubstantivity of the composition; skin bleaching (or lightening) agentsincluding but not limited to hydroquinone, kojic acid and sodiummetabisulfite; chelators and sequestrants; and aesthetic components suchas fragrances, pigments, colorings, essential oils, skin sensates,astringents, skin soothing agents, skin healing agents and the like.Non-limiting examples of these aesthetic components include panthenoland derivatives (e.g., ethyl panthenol), aloe vera, pantothenic acid andits derivatives, clove oil, menthol, camphor, eucalyptus oil, eugenol,menthyl lactate, witch hazel distillate, allantoin, bisabalol,dipotassium glycyrrhizinate and the like.

The carrier system may also comprise, when desired, a suitable gellingagent including, but not limited to, cellulose esters such ashydroxypropyl cellulose, hydroxyethyl cellulose, polyvinylpyrrolidone,carboxyvinyl polymer and the like that may be provided in any amountnecessary to thicken the composition to a desired gel consistency. Whenformulated as a gel, the base composition exhibits favorablespreadability characteristics. In addition, it remains visible on theskin surface longer, thereby instilling in the user the impression thatthe vehicle is more completely delivering its active ingredient(s).

In addition to the aforementioned ingredients, it should also be notedthat the following ingredients may also be included in embodiments ofthe inventive composition, as desired: coloring agents, fragrances,conditioners, moisturizers, surfactants, antioxidants, preservatives,etc.

Effective doses of the compositions of the present invention, fortreatment of conditions or diseases as described herein vary dependingupon many different factors, including means of administration, targetsite, physiological state of the patient, whether the patient is humanor an animal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnon-human mammals including transgenic mammals can also be treated.Treatment dosages may be titrated using routine methods known to thoseof skill in the art to optimize safety and efficacy.

The pharmaceutical compositions provided herein may include a“therapeutically effective amount.” A “therapeutically effective amount”refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic result. A therapeuticallyeffective amount of a molecule may vary according to factors such as thedisease state, age, sex, and weight of the individual, and the abilityof the molecule to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the molecule are outweighed by thetherapeutically beneficial effects.

Further provided herein are kits comprising a therapeutically effectiveamount of a tyrosine kinase inhibitor or a related compound.

As used herein, the terms “treat” and “treatment” refer to therapeutictreatment, including prophylactic or preventative measures, wherein theobject is to prevent or slow down (lessen) an undesired physiologicalchange associated with a disease or condition. Beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of the extent of a disease or condition,stabilization of a disease or condition (i.e., where the disease orcondition does not worsen), delay or slowing of the progression of adisease or condition, amelioration or palliation of the disease orcondition, and remission (whether partial or total) of the disease orcondition, whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. Those in need of treatment include those already with thedisease or condition as well as those prone to having the disease orcondition or those in which the disease or condition is to be prevented.

Examples of diseases which may be treated include a skin cancerassociated diseases, for example, pre-cancerous skin lesions, skintumors, and their associated diseases or disorders, or a cancerassociated with the tyrosine kinase mediated signaling. Particularexamples of diseases which may be treated include actinic keratosis,squamous cell carcinoma in situ like lesions, and cutaneous squamouscell carcinoma.

Methods of treating cancer may include, e.g., clearing pre-canceroustissues, inhibiting angiogenesis in the tumor, inhibiting tumor growth,inhibiting tumor migration, inhibiting proliferation or inhibitinginvasion of tumor cells.

Cancers that may be treated as described herein include those thatexpress or overexpress or are associated with the expression oroverexpression of a tyrosine kinase (e.g., SRK).

Cancers to be treated include primary tumors and secondary or metastatictumors, as well as recurrent or refractory tumors. Recurrent tumorsencompass tumors that appear to be inhibited by treatment with suchagents, but recur up to five years, sometimes up to ten years or longerafter treatment is discontinued. Refractory tumors are tumors that havefailed to respond or are resistant to treatment with one or moreconventional therapies for the particular tumor type. Refractory tumorsinclude those that are hormone-refractory; those that are refractory totreatment with one or more chemotherapeutic agents; those that arerefractory to radiation; and those that are refractory to combinationsof chemotherapy and radiation, chemotherapy and hormone therapy, orhormone therapy and radiation.

Therapy may be “first-line”, i.e., as an initial treatment in patientswho have had no prior anti-cancer treatments, either alone or incombination with other treatments; or “second-line”, as a treatment inpatients who have had one prior anti-cancer treatment regimen, eitheralone or in combination with other treatments; or as “third-line”,“fourth-line”, etc. treatments, either alone or in combination withother treatments.

Therapy may also be given to patients who have had previous treatmentswhich have been partially successful but are intolerant to theparticular treatment. Therapy may also be given as an adjuvanttreatment, i.e., to prevent reoccurrence of cancer in patients with nocurrently detectable disease or after surgical removal of tumor.

Cancers that may be treated include tumors that are not vascularized, ornot yet substantially vascularized, as well as vascularized tumors.Adult tumors/cancers and pediatric tumors/cancers are included.

More than one tyrosine kinase inhibitor (e.g., SFK inhibitor) or relatedcompound may be administered, either incorporated into the samecomposition or administered as separate compositions.

The tyrosine kinase inhibitor or related compound may be administeredalone, or in combination with one or more therapeutically effectiveagents or treatments. The other therapeutically effective agent may beconjugated to the tyrosine kinase inhibitor or related compound,incorporated into the same composition as the tyrosine kinase inhibitoror related compound, or may be administered as a separate composition.The other therapeutically agent or treatment may be administered priorto, during and/or after the administration of the tyrosine kinaseinhibitor or related compound.

In certain embodiments, the tyrosine kinase inhibitor or relatedcompound is co-administered with one or more other therapeutic agents ortreatments. In other embodiments, the tyrosine kinase inhibitor orrelated compound is administered independently from the administrationof one or more other therapeutic agents or treatments. For example, thetyrosine kinase inhibitor or related compound is administered first,followed by the administration of one or more other therapeutic agentsor treatments. Alternatively, one or more other therapeutic agents areadministered first, followed by the administration of the tyrosinekinase inhibitor or related compound. As another example, a treatment(e.g, a surgery) is carried out first, followed by the administration ofthe tyrosine kinase inhibitor or related compound.

Other therapeutically effective agents/treatments include surgery,anti-neoplastics (including chemotherapeutic agents and radiation),anti-angiogenesis agents, antibodies to other targets, small molecules,photodynamic therapy, immunotherapy, cytotoxic agents, cytokines,chemokines, growth inhibitory agents, anti-hormonal agents, kinaseinhibitors, cardioprotectants, immunostimulatory agents,immunosuppressive agents, and agents that promote proliferation ofhematological cells.

A chemotherapeutic agent according to certain embodiments of theinvention may be administered as a prodrug. The term “prodrug” refers toa precursor or derivative form of a pharmaceutically active substancethat is less cytotoxic to tumor cells compared to the parent drug and iscapable of being enzymatically activated or converted into the moreactive parent form. The prodrugs that may find use with the compositionsand methods as provided herein include but are not limited tophosphate-containing prodrugs, thiophosphate-containing prodrugs,sulfate-containing prodrugs, peptide-containing prodrugs, D-aminoacid-modified prodrugs, glycosylated prodrugs, beta-lactam-containingprodrugs, optionally substituted phenoxyacetamide-containing prodrugs oroptionally substituted phenylacetamide-containing prodrugs,5-fluorocytosine and other 5-fluorouridine prodrugs which can beconverted into the more active cytotoxic free drug. Examples ofcytotoxic drugs that can be derivatized into a prodrug form for use withcompositions and methods as provided herein include but are not limitedto any of the aforementioned chemotherapeutic agents.

The administration of the tyrosine kinase inhibitor or related compoundwith other agents and/or treatments may occur simultaneously, orseparately, via the same or different route, at the same or differenttimes. Dosage regimens may be adjusted to provide the optimum desiredresponse (e.g., a therapeutic or prophylactic response).

In one example, a single dose may be administered. In another example,several divided doses may be administered over time. In yet anotherexample, a dose may be proportionally reduced or increased as indicatedby the exigencies of the therapeutic situation. For multiple dosages,the composition may be, for example, administered three times a day,twice a day, once a day, once every two days, twice a week, weekly, onceevery two weeks, or monthly. Dosage unit form, as used herein, refers tophysically discrete units suited as unitary dosages for treatingmammalian subjects. Each unit may contain a predetermined quantity ofactive compound calculated to produce a desired therapeutic effect. Insome embodiments, the dosage unit forms of the invention are dictated byand directly dependent on the unique characteristics of the activecompound and the particular therapeutic or prophylactic effect to beachieved.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition. An ordinarily skilled physician or veterinarian can readilydetermine and prescribe the effective amount of the drug required.

“Administration” to a subject is not limited to any particular deliverysystem and may include, without limitation, topical, transdermal,parenteral (including subcutaneous, intravenous, intramedullary,intraarticular, intramuscular, or intraperitoneal injection), rectal, ororal (for example, in capsules, suspensions or tablets). Administrationmay occur in a single dose or in repeat administrations, and in any of avariety of physiologically acceptable salt forms, and/or with anacceptable pharmaceutical carrier and/or additive as part of apharmaceutical composition (described earlier). Once again,physiologically acceptable salt forms and standard pharmaceuticalformulation techniques are well known to persons skilled in the art(see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co.).

Compositions described herein (e.g., a tyrosine kinase inhibitor orrelated compound) may be administered topically as described herein. Insome embodiments, the compositions of the present invention may bedelivered to the skin using conventional dermal-type patches orarticles, wherein the active ingredients of the composition is containedwithin a laminated structure, that serves as a drug delivery device tobe affixed to the skin. In such a structure, the active ingredients ofthe composition are contained in a layer, or “reservoir”, underlying anupper backing layer. The laminated structure may contain a singlereservoir, or it may contain multiple reservoirs. In one embodiment, thereservoir comprises a polymeric matrix of a pharmaceutically acceptablecontact adhesive material that serves to affix the system to the skinduring active ingredients delivery. Examples of suitable skin contactadhesive materials include, but are not limited to, polyethylenes,polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and thelike. The particular polymeric adhesive selected will depend on theparticular active ingredients, vehicle, etc., i.e., the adhesive must becompatible with all components of the active ingredients-containingcomposition. Alternatively, the active ingredients-containing reservoirand skin contact adhesive are present as separate and distinct layers,with the adhesive underlying the reservoir which, in this case, may beeither a polymeric matrix as described above, or it may be a liquid orhydrogel reservoir, or may take some other form.

As used herein, a “composition” refers to any composition that containsa pharmaceutically effective amount of a tyrosine kinase inhibitor or arelated compound.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, carriers, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes an excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused herein includes both one and more than one such excipient.

The term “subject” includes mammals, e.g., humans, companion animals(e.g., dogs, cats, birds, and the like), farm animals (e.g., cows,sheep, pigs, horses, fowl, and the like) and laboratory animals (e.g.,rats, mice, guinea pigs, birds, and the like). In some embodiments, thesubject is human.

As used herein, the term “severity” is meant to describe the potentialof cancer to transform from a precancerous, or benign, state into amalignant state. Alternatively, or in addition, severity is meant todescribe a cancer stage or tumor grade according to an art-recognizedmethod.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per practice in the art. Alternatively, “about” with respect to thecompositions can mean plus or minus a range of up to 20%, preferably upto 10%, more preferably up to 5%.

Any reference including patents, patent applications, or scientificpublications, cited herein, are incorporated by reference in theirentirety.

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

EXAMPLES Example 1 Src-Family Tyrosine Kinases are Drivers of CutaneousNeoplasia and UVB Responses in Keratinocytes, while Srcasm, a NegativeRegulator of SFKs, is a Tumor Supressor

Src-family tyrosine kinases (SFKs) were the first oncogenes identified,and they play central roles in promoting neoplasia in most tissues. SFKsare downstream effectors of EGFR and activate the Ras-Mek-Erk pathway inhuman keratinocytes. In contrast with Src, Fyn is prominently acylatedon its N-terminus and localizes to lipid rafts, which are importantmembrane subdomains for receptor tyrosine kinase signaling. Fyn plays amajor role in keratinocyte biology as lipid rafts are critical for EGFRsignaling. Increased EGFR activity has been seen in a variety of humanepidermal cancers including actinic keratoses, squamous cell carcinoma,and Bowen' s disease.

Serial sections from 17 human AKs were analyzed for activated SFKs andSrcasm levels Immunohistochemistry demonstrated elevated SFK activityand decreased Srcasm levels in the AKs (FIG. 2). Adjacent unremarkableepidermis (Unr) demonstrated increased SFK activity in the basal celllayer with low Srcasm levels, and decreased SFK activity with higherSrcasm levels in the suprabasilar epidermis (FIG. 2). The highprevalence of increased SFK activation in these lesions suggests thatelevated SFK activity represents a key pathophysiologic feature of humanAKs and sSCCs.

To model these observations, K14-Fyn Y528F transgenic mice weregenerated. These mice spontaneously develop precancerous lesionsresembling human AKs and cSCCs in 5-8 weeks. Furthermore, these lesionsresemble the human disease at the histologic and molecular levels.Increased Fyn activity lowers p53 levels in the precancerous lesions andcSCCs, likely through a Mek/Erk/c-Jun dependent mechanism (See FIG. 1).Recent work has shown that SFKs regulate UVB-induced protein kinase D(PKD) activity in a time- and dose-dependent manner in primary murinekeratinocytes. The mechanism for activating PKD requires SFK activity.Activation of PKD in UVB-treated cells inhibits UVB-dependent apoptosisand increases the survival of keratinocytes with DNA damage, therebypromoting neoplasia. Fyn phosphorylates PKCδ, and PKCδ is an importantregulator of UVB-induced apoptosis.

Srcasm (Src-activating and signaling molecule) has been shown tonegatively regulate SFKs by targeting them for degradation in alysosomal-dependent manner. Srcasm levels are decreased in human AKs andcSCCs, and Srcasm downregulation in these lesions inversely correlateswith activation of SFKs in human lesions (FIG. 2). As discussed below,increasing Srcasm levels induces regression of established cSCCs inK14-Fyn Y528F mice suggesting that Srcasm levels in skin are criticalfor regulating cutaneous neoplasia.

Oncogenic Ras is a major driver of cSCCs. Recent in vitro data fromHaCaT cells demonstrates that Fyn is a key effector of oncogenic H-RasG12V. In these studies, oncogenic H-Ras induces Fyn mRNA >100 fold butnot Src or Yes. Fyn was required for cell invasion and motility, and Fynalso was necessary for H-Ras induced activation of focal adhesion kinase(FAK). H-Ras induces Fyn mRNA levels through an Akt-dependent mechanism.Since Fyn activates the PI-3K/Akt pathway, it may amplify its ownexpression through a positive feedback mechanism.

K14 Fyn Y528F mice demonstrate prominent epidermal hyperplasia and cSCCsin an UVB model. To determine if Fyn and Srcasm regulate UVB-inducedresponses in skin, 5-6 week old K14-Fyn Y528F, K14-Srcasm, and K14-FynY528F;K14-Srcasm transgenic mice and controls were subjected to one doseof 1200 mJ/cm² of UVB. Mice were depilated two days prior to irradiationand were biopsied at distinct sites on the back at day 0(pre-irradiation), 2, and 10; non-tumorigenic skin was biopsied inK14-Fyn Y528F mice. K14-Fyn Y528F mice demonstrated marked skinthickening by day 5; this was not seen in other cohorts (data notshown). Histologic skin sections demonstrated marked epidermalhyperplasia in K14-Fyn Y528F mice; control, K14-Srcasm, and K14-FynY528F;K14-Srcasm mice all demonstrated mild epidermal hyperplasiarelative to the day 0 biopsies (FIG. 3). The UVB-induced hyperplasia inK14-Fyn Y528F mice was associated with increased keratinocyteproliferation, and a persistent DNA damage response revealed bysubnuclear staining for phospho-H2A.X (Ser 139)(FIGS. 5 and 6).Phospho-H2A.X nuclear staining was not seen in other day 10 or any day 0biopsies. These data show that increased Fyn activity promotesUVB-induced epidermal hyperplasia and an activated DNA-damaged repairresponse. Elevated Srcasm levels in the K14-Fyn Y528F;K14-Srcasmtransgenic mice corrected the UVB-induced hyperplasia and H2A.XDNA-damage response. Keratinocyte proliferation associated withDNA-damage promotes cutaneous neoplasia and suggests that K14-Fyn Y528Fmice will have an increased susceptibility to UVB-inducedcarcinogenesis. This hypothesis was tested by chronically irradiatingK14-Fyn Y528F mice and controls. K14-Fyn Y528F mice developed multiplenew cSCCs after 7 weeks of UVB radiation while controls did not (FIG.4). K14-Fyn Y528F mice do not develop new cSCCs after 11 weeks of age.

Example 2 Effect of Small Molecule Kinase Inhibitors on PrecursorLesions and Tumors in Mice

As discussed in the previous example, K14-Fyn Y528F mice represent arobust model of cutaneous carcinogenesis which yields lesions that mimichuman precancerous lesions; this model presents a unique in vivoscreening tool to test the topical efficacy of small molecule kinaseinhibitors in preventing the formation of precancerous lesions andcutaneous SCCs.

The phenotype of K14-Fyn Y528F transgenic mice yields precancerouslesions and cutaneous SCCs that resemble their human counterparts at theclinical, histologic, and molecular levels. Characterization of theK14-Fyn Y528F model demonstrates that the formation of precancerous andcSCC lesions is directly dependent on an elevated level of activatedSFKs in keratinocytes.

Given the robust phenotype of the K14-Fyn Y528F mice, this transgenicline represents an excellent, physiologically relevant in vivo model fortesting the efficacy of topical agents in preventing precancerous andcancerous lesions.

Topical application of the small molecule EGFR inhibitor, AG1478(Caibiochem MW 315.8), has been shown to inhibit UV-induced tumorformation in transgenic models overexpressing v-Ras^(Ha).SFK-inihibitors such as PP2 (aka AG1879, Caibiochem, MW 301.8) areslightly smaller molecules and have similar physicochemical propertiesas AG1478. Therefore, AG1879 may exhibit similar cutaneous absorptioncharacteristics as AG1478, and its efficacy can be tested in inhibitingthe formation of precancerous lesions and cSCCs through topicalapplication. In addition, topical application of PP2 could be tested todetermine if the compound can cause the regression of existingprecancerous lesions and cSCCs. Additional supportive scientific datafor testing PP2 comes from studies using radio-labeled tryphostin RG14620, a non-specific tyrosine kinase inhibitor, (MW 275.1); thisradio-labeled tyrosine kinase inhibitor applied in a white petrolatumointment to rat skin demonstrated epidermal penetration. PP2 has amolecular weight (MW) of 301.8, and it meets the “500 dalton” rule forefficient penetration of the stratum corneum. For these reasons, it isimportant to test PP2 as a topical agent for treating the precancerouslesions and cSCCs in K14-Fyn Y528F mice.

Since human skin has a thicker epidermis and stratum corneum than murineskin, one may consider whether PP2 will penetrate murine skin but nothuman skin. However, there are studies demonstrating that the tyrosinekinase inhibitor, genistein, which bears structural semblance to PP2,can penetrate both murine skin and human skin reconstructs to inhibitUVB-induced signaling and DNA damage. Therefore, if we can show that PP2inhibits the formation of precancerous lesions and cSCCs in K14-FynY528F mice; it is likely that this compound or its congeners can beformulated to penetrate human skin efficiently.

Compounds:

5-fluorouracil (5-FU) applied as an ointment. Topical 5-FU is the firstline topical agent for treating AKs in humans and can be tested as thetherapeutic positive control and to validate K14-Fyn Y528F mice as amodel of cutaneous neoplasia.

AG1879 (PP2, Calbiochem) applied in DMSO. AG1879 has the chemical nameof 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolopyrimidine and it is asmall molecule inhibitor of SFKs with a molecular weight of 301.8.AG1879 is similar structurally to AG1478, an EGFR inhibitor shown topenetrate murine skin when topically applied in DMSO. AG1879 is a potentand selective inhibitor of the Src family of protein tyrosine kinases:p59^(fyn)T (IC₅₀=5 nM) and Src (IC₅₀=100 nM). Since AG1879 is slightlysmaller than AG1478, it is likely to penetrate the murine stratumcorneum as efficiently, perhaps even more efficiently than AG1478. Onecan apply 10-25 μL of a 10 μm solution to a target area using thetreatment regimens outlined below. The dose will be 1-2.5×10⁻¹¹ moles ofcompounds, which if completely absorbed into the circulation, wouldresult in a maximum systemic concentration of approximately 10 nM(average weight of adult mouse 22 g-JAX labs). If phenotypic changes areseen in non-treated areas due to systemic levels of percutaneouslyabsorbed compound, then the topical doses will be reduced in a systemicmanner.

PP3 (Calbiochem) is a negative control compound for PP2 and has thechemical name 4-Amino-7-pheny!pyrazolpyrimidine. PP3 will be applied inan analogous manner as PP2 in DMSO. PP3 has an IC₅₀=2.7 uM for EGFRwhich should not be an issue in treating K14-Fyn Y528F mice because Fynis downstream of EGFR.

Phenotypic Stages Treated with Topical Agents:

SCIS-like lesion: the squamous cell carcinoma in situ (SCIS)-likelesions are manifested by hyperkeratotic plaques (FIG. 7) that appear atapproximately 5-7 days post-natally and are present through 4 weeks ofage. The topical agents can be applied singly to individual mice inmarked treatment areas. The agent can be applied daily beginning at day5-7, and we may initially treat for 14 days. Not all hyperkeratoticplaques will be treated to determine if systemic levels ofpercutaneously absorbed PP2 affect the phenotype. Initially, ten micewill be treated with each compound; ten untreated mice will serve astreatment controls. Biopsies of treated and untreated areas can be takenat day 21.

Chemoprevention of precancerous lesions and cSCCs: K14-Fyn Y528F micedevelop precancerous lesions mimicking AKs and cutaneous SCCs between5-8 weeks of age. Application of the topical agents before 5 weeks mayprevent the formation of these lesions and demonstrate a chemopreventivepotential for the topical agents, in this study, mice may receivetopical agents to designated treatment areas beginning at 21 days andending at day 42. Each treatment group may contain ten mice, and thenon-treated control group may also comprise ten mice.

Chemoablation of pre-existing precancerous lesions and cSCCs: K14-FynY528F mice develop multiple precancerous lesions and cSCCs between 5-8weeks of age. The topical agents can be applied after the development ofthese lesions to determine if these compounds can function aschemoablative agents. After the development of precancerous lesions andcSCC, the topical agents can be applied to the lesions for 14-21 days.The treated and untreated lesions can be analyzed at the end of thetreatment course.

Evaluation of Phenotype:

The treatment groups and control mice can be analyzed as follows.

Analysis of treated and control mice: The phenotypes of each K14-FynY528F treatment group and the control group can be analyzed by countingthe number of precancerous lesions and cSCCs in treated areas andnon-treated areas of the treatment groups with those of control mice.Photographic documentation can be performed to monitor the phenotype.Phenotypic alterations can be analyzed by taking biopsies and subjectingthe tissue to the following analytical techniques:

Microscopic analysis of skin: Skin biopsies can be taken from eachtreatment group in the treated and untreated areas. Parallel biopsiescan be taken from the control group. One can characterize the histologyof precursor lesions and cSCCs from the treatment and control cohorts.The number of precursor lesions can be determined.

Western b!ot analysis: Lysates from treated and control areas can besubjected to western blot analysis to detect activated SFKs, Fyn, Notch1, NICD, Srcasm, p53, p21 and (3-actin. This can determine themechanistic effects of topical PP2 application on skin lesions.

Statistical analysis of precancerous lesions and cSCCs: The averagenumber of precancerous lesions and cSCCs can be determined for eachtreatment group and the control group. The statistical significance canbe evaluated using an independent groups T-test for the means.

Interpretations: Since the K14-Fyn Y528F phenotype depends on increasedFyn activity, the topical application of PP2 can inhibit intracellularFyn kinase activity and decrease the formation of precancerous lesionsand cSCCs. Therefore, PP2 can have chemoablative effects on precursorlesions since these lesions are unlikely to acquire other genomicmutations that might promote non-Fyn dependent, autonomous growth.

Example 3 Topical Application of the Small Molecule Tyrosine KinaseInhibitor PP2 Induces Clearance of cSCCs, Clearance of PrecancerousLesion, and Tumor Regression in Mice

To determine the effects of topical application of tyrosine kinaseinhibitors, PP2 was applied topically to a cohort of five K14-Fyn Y528Fmice with a total of 11 cSCCs. PP2 is a well characterized inhibitor ofSrc-family tyrosine kinases with a molecular weight of 301.8 daltons;therefore, this compound is smaller than 500 daltons, which is themolecular weight cut-off for penetrating the skin efficiently. Tumorsreceived approximately 25 μL of 1 mM (a 0.03% solution) PP2 daily for 30days (Monday thru Friday) and were evaluated by photography beforeinitiating treatment and every Friday for 6 weeks. As a control, acohort of 4 mice with 9 cSCCs was treated analogously with PP3, aninactive PP2 analog. The cSCCs represent keratotic tumors larger than 3x 3 mm (as measured by calipers). All PP2 treated tumors (N=11)demonstrated significant tumor regression as defined by a 75% or greaterdecrease in tumor area. None (N=9) of the PP3 treated tumorsdemonstrated 75% regression during the treatment period. These resultsare statistically significant with p<0.01. One mouse received PP2 to alarge facial cSCC and PP3 to a back cSCC; the facial tumor regressed bythe end of the 6 weeks while the back cSCC was unchanged (FIG. 7). Thesedata demonstrate that PP2 and related compounds are useful for topicallytreating cSCCs in the K14-Fyn Y528F mice. The data show that these smallmolecule kinase inhibitors can be more potent than existing agents whichare used as 0.5% to 5% formulations. It appears that systemic absorptionof the PP2 under these conditions did not affect the growth ofnon-treated tumors on the same mouse.

FIG. 8 shows that topical PP2 can induce clearance of cSCC in K14-FynY528F mice as early as 21 days. One millimolar PP2 in DMSO was topicallyapplied once a day Monday-Saturday for 20 times. Clearance of a largefacial cSCC was noted by 21 days.

To better understand the effect of topical PP2 treatment, a histologicanalysis of tissue demonstrating tumor clearance was performed. An earcovered with a cSCC as manifested by nodular masses, hyperkeratosis, andcrusting, was treated 30 times with 1 mM PP2 in DMSO (FIG. 9).Histologic features of PP2-induced clearance of cSCC in K14-Fyn Y528Fmice. One millimolar PP2 in DMSO was topically applied once a dayMonday-Friday for 30 times to a cSCC covering the dorsum of the ear.Prominent clearance of the cSCC was noted six weeks after beginningtreatment.

Prominent clearance of the tumor was noted with smoothening of the skinsurface and re-growth of hair. The histologic sections of this area showa small focus of residual cSCC adjacent to a zone of epidermalhyperplasia and mild mixed dermal inflammation. Notably absent areprominent inflammation, ulceration, or skin peeling, as might be seenwith 5-FU treatment.

As shown in FIG. 10, PP2 induces clearance of precancerous lesion inthree weeks. One millimolar PP2 in DMSO was topically applied once a dayMonday-Friday for 20 times to a small precancerous lesion on the ear andto a cSCC on the neck. Clearance of both lesions was noted by threeweeks after initiating treatment. FIG. 11 shows that PP2 induces tumorregression in five weeks.

Example 4 Topical Application of Dasatinib Induces cSCC Regression

Dasatanib (BMS-354825)(N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl[amino]-5-thiazolecarboxamide,monohydrate) is a small molecule protein kinase inhibitor where the freebase has a Molecular Weight of 488.01 daltons, and which is FDA approvedfor systemic use in the treatment of certain hematological cancers. As asmall molecule kinase inhibitor (SMKIs) (i.e., <500 daltons), dasatanibis likely to penetrate both mouse and human skin. Furthermore, as anSMKI that is FDA approved for systemic use, its toxicology profile wouldmake its approval for topical use less burdensome. Thus the efficacy oftopical dasatinib, a tyrosine kinase inhibitor, was tested on cSCCs inK14-Fyn Y528F mice. Topical 1% dasatinib ointment, formulated as shownin Table 2 below, was applied daily (Monday-Friday) to a cohort of 11mice with solitary cSCCs. A control cohort of six mice with solitarycSCCs was treated with ointment only. Topical dasatinib induced a markedregression of cSCCs in the treatment group with only 4% of the originaltumor area remaining after 5 weeks (FIGS. 10 and 11).

TABLE 2 Dasatanib Formulation Component Percentage (%) Dasatinib 1 DMSO8 Propyleneglycol 18 hydroxypropylmethylcellulose 1 Water 72

Example 5 Topical Application of BEZ235 Induces cSCC Regression

BEZ235(2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c[quinolin-1-yl[phenyl}propanenitrile)is an inhibitor of PI-3 and mTOR kinases, and these kinases areactivated in K14-Fyn Y528F cSCCs.

Phosphorylation of S6 kinase, an mTOR substrate, is elevated in thesecSCCs (FIG. 15, right panel) (right panel) as compared to unremarkabletissue (FIG. 15, left panel). Briefly, immunohistochemistry (IHC) onformalin fixed paraffin embedded (FFPE) tissue sections, 5 μm thick, wasperformed using a rabbit specific DAB detection System Kit (SpringBioscience, Pleasanton, Calif.). Slides were deparaffinized in twoxylenes and were rehydrated through a series of downgraded alcohols(100, 95, 70, and 50). Heat-mediated antigen retrieval was performedusing 10 mM Citrate Buffer, pH 6.0. To quench the endogenous peroxidase,the slides were immersed in freshly prepared 2% H₂O₂, and incubated inthe dark for 15 minutes. Slides were then washed and incubated for onehour in blocking buffer (10% normal goat serum, 1% BSA). The blockingbuffer was removed and the tissue sections were incubated overnight at4° C. with a 1:70 dilution of a rabbit anti-S6 andibody (Cell Signaling,Danvers, Mass.). The next day the tissue sections were washed in TBS.Followed by incubation with HRP conjugated secondary antibodies for 40minutes, signal was amplified with the DAB solution.

The efficacy of a 20 mM topical BEZ235 ointment, formulated as in theprevious example, was evaluated in inducing regression of cSCCs inK14-Fyn Y528F mice. Topical BEZ235 ointment was applied daily(Monday-Friday) to a cohort of 15 mice with solitary cSCCs. A controlcohort of 15 mice with solitary cSCCs was treated with ointment alone.The size range of tumors was similar in each cohort. Topical BEZ235induced a marked regression of cSCCs in the treatment group compared tocontrols (FIG. 14). In the BEZ235 cohort, 80% of tumors completelyregressed by five weeks, including many larger lesions >25mm²; incontrols, tumor shrinkage was seen in small lesions <15mm² and thiscohort has more small lesions. This data show that targeting kinasesactivated downstream of Fyn induces cSCC regression.

Example 6 Topical Application of U0126 Induces cSCC Regression

U0126 (1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene) is aselective inhibitor of both MEK1 and MEK2, both MAPK/ERK kinase familymembers, and these kinases are activated in K14-Fyn Y528F cSCCs. Theefficacy of a 1% topical U0126 ethanol or DMSO gel or ointment,formulated as in the previous examples, was evaluated for inducingregression of cSCCs in K14-Fyn Y528F mice. Topical U0126 ointment wasapplied daily (Monday-Friday) to a cohort of 8 mice with 11 cSCCs.Topical U0126 ointment was applied to each cSCC daily (Monday-Friday)for three weeks. The final measurement of cSCC sizes were conducted oneweek following the end of treatment.

TABLE 3 Treatment of cSCCs with U0126 Initial cSCC size (mm²) Final cSCCsize (mm²) 4 cSCC resolved Excluded lesion too small 9 1 9 1 14 cSCCunchanged 8 6 12 cSCC resolved 50 1 Excluded No Final MeasurementPerformed 12 cSCC resolved 1 8

As a control, 2 mice with 2 cSCCs were treated analogously with U0124(2,3-Bis-[1-amino-1-methylsulfanyl-meth-(Z)-ylidene]-succinonitrile), aninactive U0126 analog. Two of the cSCCs treated with U0124 stayed thesame size at 1 mm² Topical U0126 induces a marked regression of cSCCs inthe treatment group compared to controls of 75+/−37% compared to 0% forU0124.

Example 7 Topical Application of Selumetinib Induces cSCC Regression

Selumetinib (AZD6244)(6-(4-bromo-2-chlorophenylamino)-7-fluoro-N-(2-hydroxyethoxy)-3-methyl-3H-benzo[d]imidazole-5-carboxamide)is an MEK1 inhibitor. The efficacy of a 1% topical Selumetinib ethanolor DMSO gel or ointment, formulated as in the previous examples, isevaluated for inducing regression of cSCCs in K14-Fyn Y528F mice.Topical Selumetinib ointment is applied daily (Monday-Friday) to acohort of 15 mice with solitary cSCCs. A control cohort of 15 mice withsolitary cSCCs is treated with ointment alone. The size range of tumorsis similar in each cohort. Topical Selumetinib induces a markedregression of cSCCs in the treatment group compared to controls.

Example 8 Topical Application of Tofacitinib Induces cSCC Regression

Tofacitinib (CP-690,550) (3-[(3R,4R)-4-methyl-3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-1-yl]-3-oxopropanenitrile) is aJanus kinase (JAK) inhibitor. Tofacitinib citrate is FDA approved fororal administration in the treatment of rheumatoid arthritis. The freebase has a Molecular Weight of 312.4 daltons. The efficacy of a 1%topical Tofacitinib ethanol or DMSO gel or ointment, formulated as inthe previous examples, is evaluated for inducing regression of cSCCs inK14-Fyn Y528F mice. Topical Tofacitinib ointment is applied daily(Monday-Friday) to a cohort of 15 mice with solitary cSCCs. A controlcohort of 15 mice with solitary cSCCs is treated with ointment alone.The size range of tumors is similar in each cohort. Topical Tofacitinibinduces a marked regression of cSCCs in the treatment group compared tocontrols.

Example 9 Topical Application of Saracatinib Induces cSCC Regression

Saracatinib(N-(5-chlorobenzo[d][1,3]dioxol-4-yl)-7-(2-(4-methylpiperazin-1-yl)ethoxy)-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine)potently inhibits Src tyrosine kinase family members including Fyn. Theefficacy of a 1% topical Saracatinib ethanol or DMSO gel or ointment,formulated as in the previous examples, is evaluated for inducingregression of cSCCs in K14-Fyn Y528F mice. Topical Saracatinib ointmentis applied daily (Monday-Friday) to a cohort of 15 mice with solitarycSCCs. A control cohort of 15 mice with solitary cSCCs is treated withointment alone. The size range of tumors is similar in each cohort.Topical Saracatinib induces a marked regression of cSCCs in thetreatment group compared to controls.

Example 10 Topical Application of CEP-11981 Induces cSCC Regression

CEP-11981(13-isobutyl-4-methyl-10-(pyrimidin-2-ylamino)-4,7,8,13-tetrahydro-1H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-6(2H)-one)is a tyrosine kinase inhibitor, including Src tyrosine kinase familymembers. The efficacy of a 1% topical CEP-11981 ethanol or DMSO gel orointment, formulated as in the previous examples, is evaluated forinducing regression of cSCCs in K14-Fyn Y528F mice. Topical CEP-11981ointment is applied daily (Monday-Friday) to a cohort of 15 mice withsolitary cSCCs. A control cohort of 15 mice with solitary cSCCs istreated with ointment alone. The size range of tumors is similar in eachcohort. Topical CEP-11981 induces a marked regression of cSCCs in thetreatment group compared to controls.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications that are within the spirit and scopeof the invention, as defined by the appended claims.

What is claimed is:
 1. A method for treating a skin cancer relateddisease or disorder in a subject, the method comprising: topicallyadministering to said subject a therapeutically effective amount of aPI3K/PDK1/AKT signaling pathway inhibitor.
 2. The method of claim 1,wherein said disease or disorder is actinic keratosis.
 3. The method ofclaim 1, wherein said disease or disorder is a squamous cell carcinomain situ like lesion.
 4. The method of claim 1, wherein said disease ordisorder is associated with cutaneous squamous cell carcinoma.
 5. Themethod of claim 1, wherein said inhibitor is administered in combinationwith at least one other treatment regime selected from the groupconsisting of chemotherapy, immunotherapy, radiation therapy,photodynamic therapy, electrocautery, laser therapy, and surgery.
 6. Themethod of claim 1, wherein said subject is a human.
 7. The method ofclaim 1, wherein said inhibitor has a molecular weight of less than 500daltons.
 8. A method for treating a skin cancer related disease ordisorder in a subject, the method comprising: topically administering tosaid subject a therapeutically effective amount of a MAPK/ERK signalingpathway inhibitor.
 9. The method of claim 8, wherein said disease ordisorder is actinic keratosis.
 10. The method of claim 8, wherein saiddisease or disorder is a squamous cell carcinoma in situ like lesion.11. The method of claim 8, wherein said disease or disorder isassociated with cutaneous squamous cell carcinoma.
 12. The method ofclaim 8, wherein said inhibitor is administered in combination with atleast one other treatment regime selected from the group consisting ofchemotherapy, immunotherapy, radiation therapy, photodynamic therapy,electrocautery, laser therapy, and surgery.
 13. The method of claim 8,wherein said subject is a human.
 14. The method of claim 8, wherein saidinhibitor has a molecular weight of less than 500 daltons.
 15. A methodfor treating a skin cancer related disease or disorder in a subject, themethod comprising: topically administering to said subject atherapeutically effective amount of a JAK-STAT signaling pathwayinhibitor.
 16. The method of claim 15, wherein said disease or disorderis actinic keratosis.
 17. The method of claim 15, wherein said diseaseor disorder is a squamous cell carcinoma in situ like lesion.
 18. Themethod of claim 15, wherein said disease or disorder is associated withcutaneous squamous cell carcinoma.
 19. The method of claim 15, whereinsaid inhibitor is administered in combination with at least one othertreatment regime selected from the group consisting of chemotherapy,immunotherapy, radiation therapy, photodynamic therapy, electrocautery,laser therapy, and surgery.
 20. The method of claim 15, wherein saidsubject is a human.
 21. The method of claim 15, wherein said inhibitorhas a molecular weight of less than 500 daltons.
 23. A method fortreating a skin cancer related disease or disorder in a subject, themethod comprising: topically administering to said subject atherapeutically effective amount of a compound, wherein the compoundinhibits both PI3K and mTOR.
 24. A method for treating a skin cancerrelated disease or disorder in a subject, the method comprising:topically administering to said subject a therapeutically effectiveamount of CEP-11981.